3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert

Human intestinal morphogenesis establishes 3D epithelial microarchitecture and spatially organized crypt–villus characteristics. This unique structure is necessary to maintain intestinal homeostasis by protecting the stem cell niche in the basal crypt from exogenous microbial antigens and their metabolites. Also, intestinal villi and secretory mucus present functionally differentiated epithelial cells with a protective barrier at the intestinal mucosal surface. Thus, re-creating the 3D epithelial structure is critical to building in vitro intestine models. Notably, an organomimetic gut-on-a-chip can induce spontaneous 3D morphogenesis of an intestinal epithelium with enhanced physiological function and biomechanics. Here we provide a reproducible protocol to robustly induce intestinal morphogenesis in a microfluidic gut-on-a-chip as well as in a Transwell-embedded hybrid chip. We describe detailed methods for device fabrication, culture of Caco-2 or intestinal organoid epithelial cells in conventional setups as well as on microfluidic platforms, induction of 3D morphogenesis and characterization of established 3D epithelium using multiple imaging modalities. This protocol enables the regeneration of functional intestinal microarchitecture by controlling basolateral fluid flow within 5 d. Our in vitro morphogenesis method employs physiologically relevant shear stress and mechanical motions, and does not require complex cellular engineering or manipulation, which may be advantageous over other existing techniques. We envision that our proposed protocol may have a broad impact on biomedical research communities, providing a method to regenerate in vitro 3D intestinal epithelial layers for biomedical, clinical and pharmaceutical applications. The authors describe how to make both a gut-on-a-chip and a hybrid chip with a Transwell insert, and how to induce 3D morphogenesis of human intestinal epithelium from either Caco-2 cells or organoids using basolateral medium flow in both platforms.